CN112481133A - Culture medium for separating thraustochytrid and purification method thereof - Google Patents

Abstract

A culture medium for separating thraustochytrid and a purification method thereof are provided, the preparation of the thraustochytrid culture medium comprises the following steps: 1-5G of Glucose, 0.5-2G of Peptone, 0.5-2G of Yeast extract powder, 0.5-1G of sodium glutamate, 0.5-1G of corn steep liquor, 15-20G of Agar, 20-30G of sea salt, 0.5-1G of penicillin G, 0.5-1.5G of Streptomycin, 50-100 mg of rifampicin, 5-20 mg of Nystatin, H₂O1L, performing strain separation, morphological observation and 18S rRNA sequencing; the method can directly separate the thraustochytrid from rotten seaweed in the marine water body and the wetland, has simple culture medium components and low cost, is convenient to operate, and can be used as a separation culture method of the thraustochytrid for laboratories.

Description

Culture medium for separating thraustochytrid and purification method thereof

Technical Field

The invention relates to the field of marine microorganisms, in particular to a separation culture medium of thraustochytrid and a separation and purification method thereof.

Background

Thraustochytrids (Thraustochytrids) are heterotrophic protists of unicellular fungi in the ocean and are widely distributed in the ocean environment such as the ocean, wetlands, saline lakes, submarine sediments and the like. Currently thraustochytrids are classified as: the kingdom of algae (Stramnopila), the phylum Heterokonta (Heterokonta), the class of Glomycetes (Labyrinthulomycetes), the order thraustochytriales (Thraustochytridiales), the family thraustochytriaceae (Thraustochytridae). Thraustochytrids are not only widespread in marine ecosystems, but also play an important role in many fields. As decomposers, any substance in the sea in a rotten state (including rotten animal and plant debris, excrement of marine animals and the like) is a potential nutrient source; the thraustochytrid has the capability of independent planktonic survival in the marine environment, but is often used as parasitic microorganism which is parasitic in seaweed, seaweed or other mollusk tissues and can cause host pathogenicity, but the pathogenicity mechanism is not clear; as predators, they are also a source of both filter-feeding and clastogenic animals in food nets, and are often an important food source for crustaceans due to their high content of fatty acids in the cells. Thraustochytrid is considered a competitor of bacteria in marine ecosystems because it can degrade not only substances that are difficult to degrade but also possess huge biomass. However, there are few thraustochytrids available for laboratory culture and the media suitable for culturing them are limited.

In addition, the thraustochytrid has strong biotechnological application potential, and the cells of the thraustochytrid contain a large amount of fatty acid, which can account for more than 50% of the dry weight of the cells. Including Saturated Fatty Acids (SFAs), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA). Wherein SFA and MUFA are important raw materials for synthesizing biodiesel, and PUFA comprises docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), squalene and the like. Among them, the production of DHA by using thraustochytrium has been reported commercially. Research indicates that DHA can promote the formation of gray matter in brain, retina and heart tissue, and plays an important role in the development of the nervous and immune systems of infants. DHA can also be used as a precursor to synthesize eicosanoids, regulate in-vivo inflammatory reaction, promote blood coagulation and maintain stable blood pressure. Along with the improvement of living standard of people, people pay more and more attention to the intake of DHA. The fish oil is always the traditional source of DHA, however, the fish oil has the disadvantages of high production cost, seasonal change of oil components, scarce fish resources, heavy metal pollution, poor oxidation stability and the like, so that the synthesis of DHA by the thraustochytrid is a healthy, economic and environment-friendly way. Squalene, also a polyunsaturated terpenoid hydrocarbon, plays an important role in animals and plants, is a precursor of many steroids, including cholesterol, bile acids, hormones and vitamin D, and is widely found in nature. The oil substance produced from the liver of deep sea shark contains squalene about 80%. Squalene is also widely used as an adjuvant for pharmaceutical emulsions for the delivery of vaccines, drugs and other drugs. It can improve immune system, and thus is used as a protective agent for cancer treatment, and also as a moisturizing agent and an antioxidant for cosmetics. Shark liver oil has been the traditional source of squalene, but the random killing of these animals has caused increasing environmental problems. The accumulation of heavy metals has a number of adverse effects on human health. Therefore, the synthesis of squalene by thraustochytrium is a healthy, economic and environment-friendly way. In addition, the thraustochytrid can also secrete various extracellular enzymes, produce various extracellular polysaccharides and the like.

Disclosure of Invention

Aiming at the defects of the prior art, the culture medium for separating the thraustochytrid and the purification method thereof can directly separate the thraustochytrid from the rotten seaweed in the marine water body and the wetland, and have the advantages of simple components, low cost and convenient operation.

A culture medium formula for separating thraustochytrid comprises the following components: 1-5G of Glucose, 0.5-2G of Peptone, 0.5-2G of Yeast extract powder, 0.5-1G of sodium glutamate, 0.5-1G of corn steep liquor, 15-20G of Agar, 20-30G of sea salt, 0.5-1G of penicillin G, 0.5-1.5G of Streptomycin, 50-100 mg of rifampicin, 5-20 mg of Nystatin, H₂O 1L。

A purification method for separating thraustochytrid mainly comprises the following steps:

(1) a culture medium preparation for separating thraustochytrid comprises the following components: 1-5g of Glucose, 0.5-2g of Peptone, 0.5-2g of Yeast extract powder, 0.5-1g of sodium glutamate, 0.5-1g of corn steep liquor, 15-20g of Agar, 20-30g of sea salt, adding ultrapure water into natural pH, metering to 990ml, uniformly stirring by using a glass rod, carrying out ultrasonic treatment for 5-10min, placing in an autoclave at 121 ℃ of 115 times, and sterilizing for 15-21 min;

(2) a method for preparing antibiotic mixed liquor used for separating and purifying culture medium of thraustochytrid comprises the following steps: weighing 0.5-1G of penicillin G, 0.5-1.5G of Streptomycin, 50-100 mg of rifampicin and 5-20 mg of Nystatin, adding 10ml of ultrapure water, uniformly mixing, and oscillating by using an oscillator until the mixed solution is completely dissolved, wherein the antibiotic mixed solution needs to be prepared in situ when used; cooling the culture medium in the step (1) to 55 +/-5 ℃, pouring the antibiotic mixed solution filtered by a 0.22-micron sterile filter head, shaking uniformly, pouring the antibiotic mixed solution into a sterile culture dish, and cooling to obtain a solid culture medium for separating the thraustochytrid;

(3) separation of the strains: directly measuring 10-30ml of seawater, adding 1-2 g of pollen pini, shaking uniformly, pouring into a culture dish, culturing for 3-5 days at 27 +/-1 ℃, using an inoculating loop to pick a proper amount of pollen pini in a culture medium, directly and uniformly streaking or sucking a proper amount of mixed solution with the pollen pini in the culture medium, uniformly coating, and culturing for 2-7 days at 27 +/-1 ℃ in an incubator to obtain a prescreened strain;

(4) and (3) morphological observation: observing the form of the single colony on the flat plate by using a microscope lens of 40 multiplied by or 100 multiplied by, picking the single colony shaped like the thraustochytrid into the solid culture medium in the step (2) by using an inoculating loop under the aseptic condition for culturing, carrying out streak culture after the single colony grows out on the culture medium, and repeating the steps for 3-5 times to obtain the purified thraustochytrid;

(5) 18S rRNA sequencing: and (3) under the aseptic condition, selecting a single colony of the thraustochytrid from the plate in the step (4) by using a sterilized toothpick, carrying out PCR gene amplification, running agarose gel electrophoresis and analysis by a gel imager, and then carrying out sample DNA sequencing.

A culture medium for separating thraustochytrid and its purification method are disclosed, which can directly separate thraustochytrid from the rotten sea weed in sea water and wet land.

Drawings

FIG. 1 is a microscopic image of the isolated thraustochytrid of example 1;

FIG. 2 is a graph of the blast results from the isolation of Thraustochytrium 18Sr RNA from example 1;

FIG. 3 is a microscopic image of the isolated thraustochytrid of example 2;

FIG. 4 is a graph of the blast results from the isolation of Thraustochytrium 18Sr RNA from example 2.

Detailed Description

The invention is further illustrated by the following specific examples and figures. The examples of the present invention are for better understanding of the present invention by those skilled in the art, and do not limit the present invention in any way.

Example 1

The seawater used by the invention is collected from a seawater bath field near the Qingdao Haoshanwan in Shandong, and the collection time is 2018, 10 months and 1 day, and a culture medium for separating the thraustochytrid is used for screening and culturing:

(1) preparation of a culture medium for isolating thraustochytrid: 5g of Glucose, 2g of Peptone, 0.5g of Yeast extract powder, 0.5g of sodium glutamate, 1g of corn steep liquor, 20g of Agar, 20g of sea salt and natural pH, adding ultrapure water, metering to 990ml, uniformly stirring by using a glass rod, carrying out ultrasonic treatment for 5-10min, placing in an autoclave at 121 ℃ for 115 times, and sterilizing for 15-21 min;

(2) preparation of antibiotic mixed liquor for separating and purifying culture medium of thraustochytrid: weighing 1G of penicillin G, 1.5G of Streptomycin, 50mg of rifampicin and 5mg of Nystatin, adding 10ml of ultrapure water, uniformly mixing, and oscillating by an oscillator until the mixed solution is completely dissolved, wherein the mixed solution of the antibiotics needs to be prepared on site. Cooling the culture medium in the step (1) to 55 +/-5 ℃, pouring the antibiotic mixed solution filtered by a 0.22-micron sterile filter head, shaking uniformly, pouring the antibiotic mixed solution into a sterile culture dish, and cooling to obtain a solid culture medium for separating the thraustochytrid;

(3) directly measuring 30ml of collected seawater for separating the strains, adding 2g of pollen pini, shaking uniformly, pouring into a culture dish, culturing for 3-5 days at 27 +/-1 ℃, using an inoculating loop to pick a proper amount of pollen pini in a culture medium, directly and uniformly streaking or sucking a proper amount of mixed solution with the pollen pini in the culture medium, uniformly coating, and culturing for 2-7 days at 27 +/-1 ℃ in an incubator to obtain a primary-screened strain;

(4) and (3) morphological observation: observing the form of a single colony on a flat plate by using a microscope lens of 40 multiplied by or 100 multiplied by, picking the single colony of the thraustochytrid-like fungus into the solid culture medium in the step (2) by using an inoculating loop under the aseptic condition for culturing, carrying out streak culture after the single colony grows out on the culture medium, repeating the process for 3-5 times to obtain the purified thraustochytrid-like fungus, wherein the result is shown in figure 1;

(5) 18S rRNA sequencing: under aseptic conditions, single colony of thraustochytrid is picked from the plate in (4) by using a sterilized toothpick, and is subjected to PCR gene amplification, agarose gel electrophoresis, gel imager analysis and sample DNA sequencing, and the result of 18S rRNA sequencing analysis is shown in FIG. 2ThraustochytriumBelongs to the thraustochytrid.

Example 2

The seawater and rotten seaweed used in the invention are collected from wetland parks near the gulf of the yellow sea area, the collection time is 2018, 10 months and 1 day, and a culture medium for separating thraustochytrid is used for screening and culturing:

(1) preparation of a culture medium for isolating thraustochytrid: 1g of Glucose, 0.5g of Peptone, 2g of Yeast extract powder, 0.5g of sodium glutamate, 0.5g of corn steep liquor, 15g of Agar, 30g of sea salt, and natural pH, adding ultrapure water, then fixing the volume to 990ml, stirring uniformly by using a glass rod, carrying out ultrasonic treatment for 5-10min, placing in an autoclave at 121 ℃ for 115 times, and sterilizing for 15-21 min;

(2) preparation of antibiotic mixed liquor for separating and purifying culture medium of thraustochytrid: weighing 0.5G of penicillin G, 0.5G of Streptomycin, 100mg of rifampicin and 15mg of Nystatin, adding 10ml of ultrapure water, uniformly mixing, and oscillating by an oscillator until the mixed solution is completely dissolved, wherein the mixed solution of the antibiotics needs to be prepared as required. Cooling the culture medium in the step (1) to 55 +/-5 ℃, pouring the antibiotic mixed solution filtered by a 0.22-micron sterile filter head, shaking uniformly, pouring the antibiotic mixed solution into a sterile culture dish, and cooling to obtain a solid culture medium for separating the thraustochytrid;

(3) separating and collecting the seaweed in a rotten state, cutting the seaweed into small sections of 1-2cm by using sterilized scissors, washing the sea grass into the culture medium in the step (2) for 2-3 times by using sterile seawater, and culturing the sea grass in an incubator at the temperature of 27 +/-1 ℃ for 2-7 days to obtain a primary screening strain;

(4) and (3) morphological observation: observing the form of a single colony on a flat plate by using a microscope lens of 40 multiplied by or 100 multiplied by, picking the single colony of the thraustochytrid-like fungus into the solid culture medium in the step (2) by using an inoculating loop under the aseptic condition for culturing, carrying out streak culture after the single colony grows out on the culture medium, repeating the process for 3-5 times to obtain the purified thraustochytrid-like fungus, wherein the result is shown in a figure 3;

(5) 18S rRNA sequencing: under aseptic conditions, single colony of thraustochytrid is picked from the plate in (4) by using sterilized toothpick, and is sent to DNA sequencing after PCR gene amplification, agarose gel electrophoresis and gel imager analysis, and the result of 18S rRNA sequencing analysis is shown in FIG. 4OblongichytriumBelongs to the thraustochytrid.

Claims (2)

1. A medium for isolating thraustochytrids, comprising: the composition of the culture medium is: 1-5G of Glucose, 0.5-2G of Peptone, 0.5-2G of Yeast extract powder, 0.5-1G of sodium glutamate, 0.5-1G of corn steep liquor, 15-20G of Agar, 20-30G of sea salt, 0.5-1G of penicillin G, 0.5-1.5G of Streptomycin, 50-100 mg of rifampicin, 5-20 mg of Nystatin, H₂O 1L。

2. A method of purifying an isolated thraustochytrid culture medium according to claim 1, comprising: the method mainly comprises the following steps:

(1) preparation of a culture medium for isolating thraustochytrid: 1-5g of Glucose, 0.5-2g of Peptone, 0.5-2g of Yeast extract powder, 0.5-1g of sodium glutamate, 0.5-1g of corn steep liquor, 15-20g of Agar, 20-30g of sea salt, adding ultrapure water into natural pH, metering to 990ml, uniformly stirring by using a glass rod, carrying out ultrasonic treatment for 5-10min, placing in an autoclave at 121 ℃ of 115 times, and sterilizing for 15-21 min;

(2) preparing an antibiotic mixed solution used by a culture medium: weighing 0.5-1G of penicillin G, 0.5-1.5G of Streptomycin, 50-100 mg of rifampicin and 5-20 mg of Nystatin, adding 10ml of ultrapure water, uniformly mixing, and oscillating by using an oscillator until the mixed solution is completely dissolved, wherein the antibiotic mixed solution needs to be prepared in situ when used; cooling the culture medium in the step (1) to 55 +/-5 ℃, pouring the antibiotic mixed solution filtered by a 0.22-micron sterile filter head, shaking uniformly, pouring the antibiotic mixed solution into a sterile culture dish, and cooling to obtain a solid culture medium for separating the thraustochytrid;

(3) separation of the strains: directly measuring 10-30ml of seawater, adding 1-2 g of pollen pini, shaking uniformly, pouring into a culture dish, culturing for 3-5 days at 27 +/-1 ℃, using an inoculating loop to pick a proper amount of pollen pini in a culture medium, directly and uniformly streaking or sucking a proper amount of mixed solution with the pollen pini in the culture medium, uniformly coating, and culturing for 2-7 days at 27 +/-1 ℃ in an incubator to obtain a prescreened strain;

(4) and (3) morphological observation: observing the form of the single colony on the flat plate by using a microscope lens of 40 multiplied by or 100 multiplied by, picking the single colony shaped like the thraustochytrid into the solid culture medium in the step (2) by using an inoculating loop under the aseptic condition for culturing, carrying out streak culture after the single colony grows out on the culture medium, and repeating the steps for 3-5 times to obtain the purified thraustochytrid;

(5) 18S rRNA sequencing: and (3) under the aseptic condition, selecting a single colony of the thraustochytrid from the plate in the step (4) by using a sterilized toothpick, carrying out PCR gene amplification, running agarose gel electrophoresis and analysis by a gel imager, and then carrying out sample DNA sequencing.

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